Volume 66, Issue 2, Pages 382-389 (February 2017) The gut microbiota contributes to a mouse model of spontaneous bile duct inflammation  Elisabeth Schrumpf, Martin Kummen, Laura Valestrand, Thomas U. Greiner, Kristian Holm, Velmurugesan Arulampalam, Henrik M. Reims, John Baines, Fredrik Bäckhed, Tom H. Karlsen, Richard S. Blumberg, Johannes R. Hov, Espen Melum  Journal of Hepatology  Volume 66, Issue 2, Pages 382-389 (February 2017) DOI: 10.1016/j.jhep.2016.09.020 Copyright © 2016 European Association for the Study of the Liver Terms and Conditions

Fig. 1 NOD.c3c4 mice have a distinct global bacterial composition compared with NOD control mice. Principal coordinate plot based on unweighted UniFrac distances illustrating separation of the NOD (n=4–5) and NOD.c3c4 mice (n=5) in cecal content and mucosa (A) before (p<0.01) and (B) after rederivation into another conventional animal facility (p<0.02). (C) Intra-individual diversity measured by Shannon Diversity Index in cecal content of NOD (n=4–5) and NOD.c3c4 mice (n=5) was similar. Data in (A) and (B) compared using the anosim function in QIIME. Data in (C) are presented as mean±SEM, unpaired Student’s t test used for comparison. Journal of Hepatology 2017 66, 382-389DOI: (10.1016/j.jhep.2016.09.020) Copyright © 2016 European Association for the Study of the Liver Terms and Conditions

Fig. 2 Germ free (GF) NOD.c3c4 mice have lower liver weight and less distended common bile ducts than conventionally raised (CONV-R) NOD.c3c4 mice. (A) Total body weight and (B) liver weight as percentage of body weight of CONV-R and GF NOD.c3c4 mice at 9 (n=9 in each group) and 18weeks (w) of age (n=11 in each group). (C) Liver weight as percentage of body weight of CONV-R and GF NOD mice at 9 (GF: n=9, CONV-R: n=5) and 18 w of age (GF: n=4, CONV-R: n=5). (D) Log-transformed common bile duct dilatation (CBDD) of CONV-R and GF NOD.c3c4 mice at 9 (n=9 in each group) and 18 w of age (n=11 in each group). Data are presented as mean±SEM, unpaired Student’s t test was used for comparison. ∗p<0.05, ∗∗p<0.01, ∗∗∗p<0.001. Journal of Hepatology 2017 66, 382-389DOI: (10.1016/j.jhep.2016.09.020) Copyright © 2016 European Association for the Study of the Liver Terms and Conditions

Fig. 3 Germ free (GF) NOD.c3c4 mice have reduced inflammatory portal infiltrates compared with conventionally raised (CONV-R) NOD.c3c4 mice at 18weeks of age. (A) H&E (40×) and Sirius Red stained sections (40×, 10×) illustrating portal inflammation, bile infarcts and fibrosis. (B) Liver pathology in GF (n=11) and CONV-R (n=11) NOD.c3c4 mice scored on parameters portal inflammation, bile infarcts, fibrosis and dilatation of intrahepatic bile ducts (IHBD). Data are presented as mean±SEM, unpaired Student’s t test was used for comparison. ∗p<0.05. Journal of Hepatology 2017 66, 382-389DOI: (10.1016/j.jhep.2016.09.020) Copyright © 2016 European Association for the Study of the Liver Terms and Conditions

Fig. 4 Germ free (GF) NOD.c3c4 mice have less CD3, Ly6G and Mac-2 positive cells around their bile ducts compared with conventionally raised (CONV-R) NOD.c3c4 mice. (A) CD3, Ly6G and Mac-2 positive cells around bile ducts (indicated by arrows) in 18weeks old CONV-R (n=11) and GF (n=11) NOD.c3c4 mice (40×). (B) Mean CD3/Ly6G/Mac-2 positive cell count from six different areas with bile ducts of CONV-R and GF NOD.c3c4 mice. CD3, Ly6G and Mac-2 are makers of T cells, neutrophils and macrophages, respectively. Data are presented as mean±SEM, for CD3+ count unpaired Student’s t test was used, for Ly6G+ and Mac-2+ count Mann-Whitney U test was used for comparison due to a non-normal data distribution. ∗p<0.05, ∗∗∗p<0.001. Journal of Hepatology 2017 66, 382-389DOI: (10.1016/j.jhep.2016.09.020) Copyright © 2016 European Association for the Study of the Liver Terms and Conditions

Fig. 5 Antibiotic treated (AB) NOD.c3c4 mice have lower liver weight and less biliary infarcts compared with NOD.c3c4 mice receiving normal drinking water (control). (A) Liver weight as percentage of body weight of AB NOD.c3c4 (n=19) and control NOD.c3c4 (n=19) mice. AB NOD.c3c4 mice received treatment for 4weeks from weaning. (B) Log-transformed common bile duct dilatation (CBDD) and (C) alanine transaminase (ALT) levels measured in serum of AB and control NOD.c3c4 mice. (D) Liver pathology scored on parameters portal inflammation, bile infarcts, fibrosis and dilatation of intrahepatic bile ducts (IHBD). The data represents results from two pooled, independent experiments. Data are presented as mean±SEM, unpaired Student’s t test was used for comparison. ∗p<0.05, ∗∗∗p<0.001. Journal of Hepatology 2017 66, 382-389DOI: (10.1016/j.jhep.2016.09.020) Copyright © 2016 European Association for the Study of the Liver Terms and Conditions

Journal of Hepatology 2017 66, 382-389DOI: (10. 1016/j. jhep. 2016. 09 Copyright © 2016 European Association for the Study of the Liver Terms and Conditions